This invention relates to novel bicyclic pyrimidine derivatives that are useful in the treatment of hyperproliferative diseases, such as cancers, in mammals. This invention also relates to a method of using such compounds in the treatment of hyperproliferative diseases in mammals, especially humans, and to pharmaceutical compositions containing such compounds.
Compounds that are useful in the treatment of hyperproliferative diseases are also disclosed in the following co-pending patent applications: PCT international patent application number PCT/IB97/100675 (filed Jun. 11, 1997), United States provisional patent application No. 60/028881 (filed Oct. 17, 1996), PCT international patent application number PCT/IB97/00584 (filed May 22, 1997), U.S. patent application Ser. No. 08/653,786 (filed May 28, 1996), PCT international patent application publication number WO 96/40142 (published Dec. 19, 1996), and PCT international patent application publication number WO 95/23141 (published Aug. 31, 1995). Each of the foregoing United States and PCT international patent applications is incorporated herein by reference in its entirety.
It is known that a cell may become cancerous by virtue of the transformation of a portion of its DNA into an oncogene (i.e., a gene that upon activation leads to the formation of malignant tumor cells). Many oncogenes encode proteins which are aberrant tyrosine kinases capable of causing cell transformation. Alternatively, the overexpression of a normal proto-oncogenic tyrosine kinase may also result in proliferative disorders, sometimes resulting in a malignant phenotype.
Receptor tyrosine kinases are large enzymes that span the cell membrane and possess an extracellular binding domain for growth factors such as epidermal growth factor, a transmembrane domain, and an intracellular portion that functions as a kinase to phosphorylate specific tyrosine residue in proteins and hence to influence cell proliferation. It is known that such kinases are often aberrantly expressed in common human cancers such as breast cancer, gastrointestinal cancer such as colon, rectal or stomach cancer, leukemia, and ovarian, bronchial or pancreatic cancer. It has also been shown that epidermal growth factor receptor (EGFR), which possesses tyrosine kinase activity, is mutated or overexpressed in many human cancers such as brain, lung, squamous cell, bladder, gastric, breast, head and neck, oesophageal, gynecological and thyroid cancers. Thus, it is believed that inhibitors of receptor tyrosine kinases, such as the compounds of the present invention, are useful as selective inhibitors of the growth of mammalian cancer cells.
It has also been shown that EGFR inhibitors may be useful in the treatment of pancreatitis and kidney disease (such as proliferative glomerulonephritis and diabetes-induced renal disease), and may reduce successful blastocyte implantation and therefore may be useful as a contraceptive. See PCT international application publication number WO 95/19970 (published Jul. 27, 1995).
It is known that polypeptide growth factors such as vascular endothelial growth factor (VEGF) having a high affinity to the human kinase insert-domain-containing receptor (KDR) or the murine fetal liver kinase 1 (FLK-1) receptor have been associated with the proliferation of endothelial cells and more particularly vasculogenesis and angiogenesis. See PCT international application publication number WO 95/21613 (published Aug. 17, 1995). Agents, such as the compounds of the present invention, that are capable of binding to or modulating the KDR/FLK-1 receptor may be used to treat disorders related to vasculogenesis or angiogenesis such as diabetes, diabetic retinopathy, hemangioma, glioma, melanoma, Kaposi""s sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
The present invention relates to compounds of the formula 
and to pharmaceutically acceptable salts thereof, wherein;
Z is a group of the formula 
wherein n is an integer from 0 to 2 and p is an integer from 0 to 3;
R1 is H, C1-C6 alkyl or xe2x80x94C(O)(C1-C6 alkyl);
R2 is phenyl or 1H-indazol-5-yl, wherein said groups are optionally substituted by 1 to 3 R5 substituents, or R2 is a group of the formula (Ii) or (Ij) 
wherein p is an integer from 0 to 3 and n is an integer from 0 to 2;
or R1 and R2 are taken together to form a group of the formula (Ik) 
wherein the dashed line indicates a single or double bond and m is an integer from 0 to 4;
each R3 is independently H, xe2x80x94C(O)OR9, or C1-C6 alkyl wherein said alkyl is optionally substituted by halo, xe2x80x94OR9, xe2x80x94NR9R10 or xe2x80x94C(O)OR9;
R4 is R3 xe2x80x94OR9, or xe2x80x94NR9R10;
each R5 is independently halo, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6, alkynyl, xe2x80x94OR9, xe2x80x94NR9R10, nitro, or C6-C10 aryl wherein said alkyl, alkenyl, alkynyl and aryl R5 groups are optionally substituted by 1 to 3 substituents independently selected from halo, nitro, C1-C4 alkyl and xe2x80x94OR9;
R6 and R7 are independently H or R5;
R8 is H, xe2x80x94SO2(C6-C10 aryl), xe2x80x94(CH2)q(5-10 membered heterocyclyl), C2-C6 alkenyl, C1-C6 alkyl, xe2x80x94(CH2)q(CH2)q(C1-C6 alkoxy), xe2x80x94(CH2)q(C1-C6 alkoxy), xe2x80x94C(O)(C1-C6 alkoxy), or xe2x80x94SO2(C1-C4 alkyl) wherein each q is independently an integer from 2 to 4;
each R9 and R10 is independently H or C1-C6 alkyl; and,
R11 is trifluoromethyl, halo, nitro, xe2x80x94OR9, xe2x80x94NR9R10, cyano, C1-C4 alkyl, xe2x80x94S(O)xR9 wherein x is an integer from 0 to 2,xe2x80x94C(O)OR9, xe2x80x94OC(O)(C1-C4 alkyl), xe2x80x94C(O)NR9R10), xe2x80x94NR9C(O)(C1-C4 alkyl), xe2x80x94C(O)NHSO2(C1-C4 alkyl), xe2x80x94NHCH2C(O)NR9R10, xe2x80x94NHC(O)(C1-C4 alkoxy), xe2x80x94NHOC(O)(C1-C4 alkyl), xe2x80x94NR9OR10, anilino, pyrrolidinyl, piperidinyl, azido, guanidino, morpholino, phenyl, xe2x80x94C(O)(C1-C6 alkyl), benzenesulfonyl, allyl, thiophenyl, piperazinyl, 4-(C1-C4 alkyl)-piperazinyl, phenyftnio, benzenesulphonamido, 2-oxopyrrolidin-1-yl, 2,5dioxopyrrolidin-1-yl, phenoxy, benzoyloxy, benzoylamino, xe2x80x94(CH2)wO(CH2)vOR9, xe2x80x94O(CH2)wO(CH2)vOR9, xe2x80x94O(CH2)wC(O)OR9, xe2x80x94O(CH2)wC(O)NR9R10, xe2x80x94(CH2)wS(CH2)vOR9,xe2x80x94NH(CH2)vO(C1-C4 alkyl), xe2x80x94NH(CH2)w(C6-C10 aryl), xe2x80x94NHC(O)(CH2)w(C1-C4 alkoxy), or xe2x80x94O(CH2)w(C6-C10 aryl), wherein w is an integer from 1 to 4 and v is an integer from 2 to 4, and wherein the alkyl, heterocyclic, and aryl moieties of the foregoing R11 groups are optionally substituted by 1 or 2 substituents independently selected from the group consisting of halo, C1-C4 alkyl, xe2x80x94OR9,xe2x80x94NR9R10, xe2x80x94C(O)OR9, xe2x80x94OC(O)(C1xe2x80x94C4 alkyl), xe2x80x94C(O)NR9R10, xe2x80x94NHC(O)(C1-C4 alkyl), nitro, imidazolyl, piperidino, morpholino, and piperazinyl;
with the proviso that where Z is a group of the formula (Ie), and R2 is phenyl, then said phenyl is substituted by 1 to 3 substituents independently selected from C1-C6 alkyl, C2-C6 alkynyl and halo, and one of R4 and R7 is halo or H and the other is as defined above; and,
with the further proviso that where Z is a group of the formula (Ia), (Ib), (Ic) or (Id), and R2 is phenyl, then said phenyl is substituted by C2-C6 alkynyl.
Preferred compounds of formula I include those wherein R2 is optionally substituted phenyl. More preferred are those compounds of formula I wherein R2 is phenyl substituted by C2-C6 alkynyl, in particular ethynyl.
Other preferred compounds of formula I include those wherein Z is a pyrrolo moiety of formula (Ie) or (If).
Other preferred compounds of formula I include those wherein R1 and R2 are taken together to form an indole or indoline moiety of formula (1k),
Specific preferred compounds of formula I include the following:
4-(6-Chloro-2,3dihydro-indol-1-yl)-7H-pyrrolo[2,3-d]pyrimidine;
4-(6-Methyl-2,3dihydro-indol-1-yl)-7H-pyrrolo[2,3-d]pyrimidine;
4 (6-Chloro-5-fluoro-2,3dihydryo-indol-1-yl)-7H-pyrrolo[2,3]pyrimidine:
1-(4-m-Tolylamino-pyrrolo[2,3-d]pyrimidin-7-yl)-ethanone;
4-(6Chloro-2,3-dihydro-indol-1-yl)-pyrido[3,4-d]pyrimidine;
4-(6-Bromo-5chloro-2,3dihydro-indol-1-yl)-pyrido[3,4-d]pyrimidine;
4-(6-Fluoro5chloro-2,3-dihydro-indol-1-yl)-pyrido[3,4-d]pyrimidine;
4(6lodo-2,3-dihydro-indol-1-yl)-pyrido[3,4-d]pyrimidine;
(7-Benzenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-(3ethynyl-phenyl)-amine;
4-(6-Chloro-2,3-dihydro-indol-1-yl)-5H-pyrrolo[3,2-d]pyrimidin-6-ol;
(3-Ethynyl-phenyl)-[7(2morpholin-4-yl-ethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-amine;
(3-Ethynyl-phenyl)-[7-(2-methoxy-ethyl)-7H-pyrrolo[2,3-d]pyrmidin-4-yl]-amine;
(3Ethynyl-phenyl)-{7[2-(2-methoxy-ethoxy)ethyl]-7H-pyrrolo[2,3-d]pyrmidin4-yl}amine;
(7-Allyl-pyrrolo[2,3-d]pyrimidin-4-yl)-(3ethynyl-phenyl)-amine;
N-(5lodo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-m-tolyl-acetamide;
4-(6-Chloro-2,3dihydro-indol-1-yl)6-methyl-pyrido[3,4-d]pyrimidine;
4-(6-Bromo-5-fluoro-2,3dihydro-indol-1-yl)6methyl-pyrido[3,4-d]pyrimidine;
4-(6-Chloro5-fluoro-2,3dihydro-indol-1-yl)-6-methyl-pyrido[3,4-d]pyrimidine;
4-(6-lodo-2,3dihydro-indol-1-yl)-6-methyl-pyrido[3,4]pyrimidine;
4-(4-Bromo-7-methyl-2,3-dihydro-indol-1-yl)6-methyl-pyrido[3,4-d]pyrimidine;
4-(6-Bromo-7-methyl-2,dihydro-indol-1-yl)-6-methyl-pyrido[3,4-d]pyrimidine;
4-(6,7-Dimethyl-2,3dihydro-indol-1-yl)pyrido[3,4-d]pyrimidine;
(3-Ethynyl-phenyl)pyrido[3,4-d]pyrimidin-4-yl-amine;
Benzo[b]thiophen-5yl-pyrido[3,4-d]pyrimidin4-yl-amine;
(3-Ethynyl-phenyl)-(5-tolyl-7H-pyrrolo[2,3-d]pyrimidin4-yl)-amine;
(3-Ethynyl-phenyl)-(5-thiophen-2-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amine;
(3-Ethynyl-phenyl)-[5-(4-methoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-amine;
(3-Ethynyl-phenyl)-[5-(3nitro-phenyl)7H-pyrrolo[2,3-d]pyrimidin-4-yl]-amine;
[5-(4-chloro-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-(3-ethynyl-phenyl)-amine;
(3-Bromo-phenyl)-(6-bromo-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amine; and the pharmaceutically acceptable salts of the foregoing compounds.
The invention also relates to a pharmaceutical composition for the treatment of a hyperproliferative disorder in a mammal which comprises a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In one embodiment, said pharmaceutical composition is for the treatment of cancer such as brain, lung, squamous cell, bladder, gastric, pancreatic, breast, head, neck, renal, kidney, ovarian, prostate, colorectal, oesophageal, gynecological or thyroid cancer. In another embodiment, said pharmaceutical composition is for the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e.g., psoriasis) or prostate (e.g., benign prostatic hypertropy (BPH)).
The invention also relates to a pharmaceutical composition for the treatment of a hyperproliferative disorder in a mammal which comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, in combination with an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, and anti-androgens, and a pharmaceutically acceptable carrier.
The invention also relates to a pharmaceutical composition for the treatment of pancreatitis or kidney disease (including proliferative glomerulonephritis and diabetes-induced renal disease) in a mammal which comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The invention also relates to a pharmaceutical composition for the prevention of blastocyte implantation in a mammal which comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The invention also relates to a pharmaceutical composition for treating a disease related to vasculogenesis or angiogenesis in a mammal which comprises a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In one embodiment, said pharmaceutical composition is for treating a disease selected from the group consisting of diabetes, diabetic retinopathy, hemangioma, glioma, melanoma, Kaposi""s sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
The invention also relates to a method of treating a hyperproliferative disorder in a mammal which comprises administering to said mammal a therapeutically effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof. In one embodiment, said method relates to the treatment of cancer such as brain, squamous cell, bladder, gastric, pancreatic, breast, head, neck, oesophageal, prostate, colorectal, lung, renal, kidney, ovarian, gynecological or thyroid cancer. In another embodiment, said method relates to the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e.g., psoriasis) or prostate (e.g., benign prostatic hypertropy (BPH)).
The invention also relates to a method for the treatment of a hyperproliferative disorder in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, and anti-androgens.
The invention also relates to a method of treating pancreatitis or kidney disease in a mammal which comprises administering to said mammal a therapeutically effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof.
The invention also relates to a method of preventing blastocyte implantation in a mammal which comprises administering to said mammal a therapeutically effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof.
The invention also relates to a method of treating diseases related to vasculogenesis or angiogenesis in a mammal which comprises administering to said mammal an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. In one embodiment, said method is for treating a disease selected from the group consisting of diabetes, diabetic retinopathy, hemangioma, glioma, melanoma, Kaposi""s sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
Patients that can be treated with compounds of the formula I, and the pharmaceutically acceptable salts of said compounds, according to the methods of this invention include, for example, patients that have been diagnosed as having psoriasis, BPH, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin""s disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphonas, cancer of the bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e.g., primary CNS lymphona, spinal axis tumors, brain stem gliomas or pituitary adenomas).
The term xe2x80x9chaloxe2x80x9d, as used herein, unless otherwise indicated, means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, cyclic or branched moieties. It is understood that for cyclic moieties at least three carbon atoms are required in said alkyl group.
The term xe2x80x9calkoxyxe2x80x9d, as used herein, unless otherwise indicated, includes O-alkyl groups wherein xe2x80x9calkylxe2x80x9d, is as defined above.
The term xe2x80x9carylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl.
The term xe2x80x9c5-10 membered heterocyclylxe2x80x9d, as used herein, unless otherwise indicated, includes aromatic and non-aromatic heterocyclic groups containing one or more heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 5-10 atoms in its ring system. The heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or more oxo moieties. An example of a 5 membered heterocyclic group is thiazolyl, and an example of a 10 membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, piperidino, morpholino, thiomorpholino and piperazinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl and thiazolyl. Heterocyclic groups having a fused benzene ring include benzimidazolyl.
The phrase xe2x80x9cpharmaceutically acceptable salt(s)xe2x80x9d, as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of formula I. The compounds of formula I that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of formula I are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate)) salts.
Those compounds of the formula I that are acidic in nature, are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts and particularly, the sodium and potassium salts.
Certain compounds of formula I may have asymmetric centers and therefore exist in different enantiomeric forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of formula I and mixtures thereof. The compounds of formula I may also exist as tautomers. This invention relates to the use of all such tautomers and mixtures thereof.
The preparation of the compounds of the present invention is illustrated in the following Schemes 1 and 2. 
The compounds of the present invention are readily prepared according to synthetic methods familiar to those skilled in the art. Such methods are disclosed in PCT international patent application publication number WO 96/40142 (published Dec. 19, 1996), referred to above, and in PCT international application publication numbers WO 95/19774 (published Jul. 27, 1995) and WO 95/19970 (published Jul. 27, 1995), each of which is incorporated herein by reference.
Scheme 1 illustrates the coupling of the bicyclic compound of formula II with the amine of formula III to provide the compound of formula I. In the compounds of formulas II and III, X is hydroxy or chloro and Z. R1 and R2 are as defined above. In general, the compound of formula II is coupled with the amine of formula III in a solvent, such as a C1-C6 alcohol, dimethylformamide (DMF), N-methylpyrrolidin-2-one (NMP), chloroform, acetonitrile, tetrahydrofuran (THF), dimethylsulfoxide (DMSO), 1,4-dioxane or pyridine, optionally in the presence of a base, such as pyridine or triethylamine, and optionally in the presence of pyridine hydrochloride as a catalyst, under an inert atmosphere, such as dry nitrogen gas, at a temperature of from ambient to reflux temperature, preferably 80-125xc2x0 C., for a period of about 2 hours to 72 hours.
The bicyclic compound of formula II is prepared according to synthetic methods known to those skilled in the art. Such methods are disclosed in WO 95/19774 and WO 95/19970, referred to above. Other methods of preparing the compounds of formula II are also disclosed in PCT international application publication number WO 92/12718 (published Aug. 6, 1992), A. Petric et al., Fur Chemie 114, 615-624 (1983) and Nucleic Acids Research, v. 12, no. 2 (1984). Where the compound of formula III is an optionally substituted indole or indoline moiety, such compounds can be prepared according to one or more methods known to those skilled in the art. Such methods are described in PCT international patent application publication number WO 95/23141, referred to above, and in W. C. Sumpter and F. M. Miller, xe2x80x9cHeterocylic Compounds with Indole and Carbazole Systems,xe2x80x9d in volume 8 of xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d, Interscience Publishers Inc., New York (1954). Optional substituents can be included as appropriate before or after the coupling step illustrated in Scheme 1. Prior to the coupling step, primary and secondary amino moieties (other than the amine of formula III) are preferably protected using a nitrogen protecting group known to those skilled in the art. Such protecting groups and their use are described in T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d Second Edition, John Wiley and Sons, New York, 1991.
Scheme 2 illustrates the cyclization of the compound of formula IV (wherein Me means methyl) to provide the compound of formula V. This illustrates the introduction of the Z moiety in the compound of formula I after the amino (xe2x80x94NR1R2) moiety has been coupled with the pyrimidine ring. .While Scheme 2 illustrates the formation of a pyrrolo[3,2-d]pyrimidine ring system, other ring systems can be made by one or more analogous methods disclosed in WO 90/19774, referred to above. In Scheme 2, the compound of formula IV, in an alcoholic solvent, such as ethanol, is catalytically hydrogenated using an appropriate catalyst, such as 10% palladium on carbon, under an H2 atmosphere at ambient temperature for a period of about three hours to provide the compound of formula V. Optional substituents can be introduced into the compound of formula V, as appropriate, according to methods known to those skilled in the art, to provide the desired compound of formula I.
The compounds of the present invention may have asymmetric carbon atoms. Such diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known to those skilled in the art, for example, by chromatography or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixtures into a diastereomric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomer mixtures and pure enantiomers are considered as part of the invention.
The compounds of formula I that are basic in nature are capable of forming a wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceptable for administration to animals, it is often desirable in practice to initially isolate the compound of formula I from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is readily obtained. The desired acid salt can also be precipitated from a solution of the free base in an organic solvent by adding to the solution an appropriate mineral or organic acid.
Those compounds of the formula I that are acidic in nature, are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline-earth metal salts and particularly, the sodium and potassium salts. These salts are all prepared by conventional techniques. The chemical bases which are used as reagents to prepare the pharmaceutically acceptable base salts of this invention are those which form non-toxic base salts with the acidic compounds of formula I. Such non-toxic base salts include those derived from such pharmacologically acceptable cations as sodium, potassium calcium and magnesium, etc. These salts can easily be prepared by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations, and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum yields of the desired final product.
The active compounds of this invention are potent inhibitors of the erbB family of oncogenic and protooncogenic protein tyrosine kinases such as epidermal growth factor receptor (EGFR), erbB2, HER3, or HER4 and thus are all adapted to therapeutic use as antiproliferative agents (e.g., anticancer) in mammals, particularly in humans. In particular, the compounds of this invention are useful in the prevention and treatment of a variety of human hyperproliferative disorders such as malignant and benign tumors of the liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, glioblastomas, head and neck, and other hyperplastic conditions such as benign hyperplasia of the skin (e.g., psoriasis) and benign hyperplasia of the prostate (e.g., BPH). It is, in addition, expected that a compound of the present invention may possess activity against a range of leukemias and lymphoid malignancies.
The active compounds may also be useful in the treatment of additional disorders in which aberrant expression ligand/receptor interactions or activation or signalling events related to various protein tyrosine kinases, are involved. Such disorders may include those of neuronal, glial, astrocytal, hypothalamic, and other glandular, macrophagal, epithelial, stromal, and blastocoelic nature in which aberrant function, expression, activation or signalling of the erbB tyrosine kinases are involved. In addition, compounds of formula I may have therapeutic utility in inflammatory, angiogenic and immunologic disorders involving both identified and as yet unidentified tyrosine kinases that are inhibited by compounds of the formula I.
The in vitro activity of the active compounds in inhibiting the receptor tyrosine kinase (and thus subsequent proliferative response, e.g., cancer) may be determined by the following procedure.
Activity of the active compounds, in vitro, can be determined by the amount of inhibition of the phosphorylation of an exogenous substrate (e.g., Lys3-Gastrin or polyGluTyr (4:1) random copolymer (I. Posner et al., J. Biol. Chem. 267 (29), 20638-47 (1992)) on tyrosine by epidermal growth factor receptor kinase by a test compound relative to a control. Affinity purified, soluble human EGF receptor (96 ng) is obtained according to the procedure in G. N. Gill, W. Weber, Methods in Enzymology 146, 82-88 (1987) from A431 cells (American Type Culture Collection, Rockville, Md.) and preincubated in a microfuge tube with EGF (2,xcexcg/ml) in phosphorylation buffer +vanadate (PBV: 50 mM HEPES, pH 7.4; 125 mM NaCl; 24 mM MgCl2; 100 xcexcM sodium orthovanadate), in a total volume of 10 xcexcl, for 20-30 minutes at room temperature. The test compound, dissolved in dimethylsulfoxide (DMSO), is diluted in PBV, and 10 xcexcl, is mixed with the EGF receptor IEGF mix, and incubated for 10-30 minutes at 30xc2x0 C. The phosphorylation reaction is initiated by addition of 20 xcexcl33P-ATP/substrate mix (120 xcexcM Lys3-Gastrin (sequence in single letter code for amino acids, KKKGPWLEEEEEAYGWLDF), 50 mM Hepes pH 7.4, 40 xcexcM ATP, 2 xcexcCi xcex3-[33P]-ATP) to the EGFr/EGF mix and incubated for 20 minutes at room temperature. The reaction is stopped by addition of 10 xcexcl stop solution (0.5 M EDTA, pH 8; 2 mM ATP) and 6 xcexcl 2N HCl. The tubes are centrifuged at 14,000 RPM, 4xc2x0 C., for 10 minutes. 35 xcexcl of supernatant from each tube is pipetted onto a 2.5 cm circle of Whatman P81 paper, bulk washed four times in 5% acetic acid, 1 liter per wash, and then air dried. This results in the binding of substrate to the paper with loss of free ATP on washing. The [33P] incorporated is measured by liquid scintillation counting. Incorporation in the absence of substrate (e.g., lys3-gastrin) is subtracted from all values as a background and percent inhibition is calculated relative to controls without test compound present.
Such assays, carried out with a range of doses of test compounds, allow the determination of an approximate IC50 value for the in vitro inhibition of EGFR kinase activity. The compounds of the formula I that were tested using the procedure described above exhibited IC50 values in the range of 0.0001-30 xcexcM.
Activity of the active compounds, in vivo, can be determined by the amount of inhibition of tumor growth by a test compound relative to a control. The tumor growth inhibitory effects of various compounds are measured according to the methods of Corbett T. H., et al. xe2x80x9cTumor Induction Relationships in Development of Transplantable Cancers of the Colon in Mice for Chemotherapy Assays, with a Note on Carcinogen Structurexe2x80x9d, Cancer Res., 35, 2434-2439 (1975) and Corbett, T. H., et al., xe2x80x9cA Mouse Colon-tumor Model for Experimental Therapyxe2x80x9d, Cancer Chemother. Rep. (Part 2)xe2x80x9d, 5, 169-186 (1975), with slight modifications. Tumors are induced in the left flank by s.c. injection of 1xc3x97106 log phase cultured tumor cells (human MDA-MB-468 breast or human HN5 head and neck carcinoma cells) suspended in 0.10 ml RPMI 1640. After sufficient time has elapsed for the tumors to become palpable (2-3 mm in diameter) the test animals (athymic mice) are treated with active compound (formulated by dissolution in DMSO typically at a concentration of 50 to 100 mg/mL followed by 1:9 dilution into saline or, alternatively, 1:9 dilution into 0.1% Pluronic(copyright) P105 in 0.9% saline) by the intraperitoneal (ip) or oral (po) routes of administration twice daily i.e., every 12 hours) for 5 consecutive days. In order to determine an anti-tumor effect, the tumor is measured in millimeters with Vernier calipers across two diameters and the tumor size (mg) is calculated using the formula: Tumor weight=(lengthxc3x97[width]2)/2, according to the methods of Geran, R. I., et al. xe2x80x9cProtocols for Screening Chemical Agents and Natural Products Against Animal Tumors and Other Biological Systemsxe2x80x9d, Third Edition, Cancer Chemother. Rep., 3, 1-104 (1972). Results are expressed as percent inhibition, according to the formula: Inhibition (%)=(TuWcontrolxe2x88x92TuWtest)/TuWcontrolxc3x97100%. The flank site of tumor implantation provides reproducible dose/response effects for a variety of chemotherapeutic agents, and the method of measurement (tumor diameter) is a reliable method for assessing tumor growth rates. The title compounds of the experimental examples of this case that are compounds of the formula I all exhibited, when tested in the above assay, percent inhibition values greater than 50% at 10 xcexcM.
Other methods of assessing the activity of the compounds of the present invention are referred to in PCT international application publication number WO 95/21613 (published Aug. 17, 1995) which incorporated herein by reference.
Administration of the active compounds can be effected by any method that enables delivery of the compounds to the site of action (e.g., cancer cells). These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical administration, etc.
The amount of the active compound administered will be dependent on the subject being treated, the severity of the disorder or condition, the rate of administration and the judgement of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 to about 35mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to about 7 g/day, preferably about 0.2,to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.
The active compound may be applied as a sole therapy or may involve one or more other anti-tumour substances, for example those selected from, for example, mitotic inhibitors, for, example vinblastine; alkylating agents, for example cis-platin, carboplatin and cyclophosphamide; anti-metabolites, for example 5-fluorouracil, cytosine arabinoside and hydroxyurea, or, for example, one of the preferred anti-metabolites disclosed in European Patent Application No. 239362 such as N-(5[N-(3,4-dihydro-2-methyl-4-oxoquinazolin6-ylmethyl)-N-methylamino]2-thenoyl)-L-glutamic acid; intercalating antibiotics, for example adriamycin and bleomycin; enzymes, for example interferon; and anti-hormones, for example anti-estrogens such as Nolvadex(trademark) (tamoxifen) or, for example anti-androgens such as Casodex(trademark) (4-cyano-3-(4-fluomphenylsulphonyl)-2hydroxy-2methyl-3xe2x80x2-(trifluoromethyl)propionanilide). Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.
The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.
Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. The pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus for oral administration, tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Preferred materials, therefor, include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions, or elixirs are desired for oral administration the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in this art. For examples, see Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).
The following examples illustrate the preparation of the compounds of the invention. In the following examples, xe2x80x9cMexe2x80x9d means methyl and xe2x80x9cEtxe2x80x9d means ethyl.